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1. In tro duc tion
Denatonium ben zo ate, Bitrex, is a sub stance that
has been used all over the world for many years to de -
na ture in dus trial al co hol. The com mon name for this
chem i cal, denatonium, al ludes to this ap pli ca tion. It is
char ac ter ised by a very in ten sive, bit ter taste, which
means that a very small ad di tion makes prod ucts un -
suit able for con sump tion. There fore, it is an ideal ad di -
tive to pre vent peo ple from ac ci den tally in gest ing
liq uids such as de ter gents, dis in fec tants, clean ing flu -
ids, and gar den and hor ti cul tural prod ucts. Dif fer ences
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
ASSESSMENT OF THE EFECTIVENESS OF DENATONIUM
BENZOATE (BITREX) REMOVAL FROM DENATURED SPIRIT
PREPARATIONS USING SODIUM HYPOCHLORITE
Dariusz ZUBA, Czes³awa ŒWIEGODA, Bogumi³a BYRSKA, Wojciech LECHOWICZ
In sti tute of Fo ren sic Re search, Krakow
Ab stract
The aim of the study was to as sess the ef fec tive ness of pu ri fi ca tion of spirit prep a ra tions de na tured by Bitrex by means of so dium
hyphochlorite. The sub ject mat ter was cho sen fol low ing anal y sis of ev i den tial ma te rial sub mit ted to the In sti tute, which in di cated
that such a pro cess might be ap plied by com pa nies trad ing in prod ucts pre pared on the ba sis of al co hol, e.g. wind screen wash ers,
dis in fec tants, al co holic sol vents, or bar be cue light ing fluid. High-per for mance liq uid chro ma tog ra phy (HPLC) was used in the
anal y sis and the ob tained re sults were con firmed by means of mass spec trom e try (MS). The ad di tion of so dium hypochlorite to
spirit prep a ra tions causes the for ma tion of a white pre cip i ta tion, the amount of which in creases to gether with an in crease in the
amount of so dium hypochlorite added to the so lu tion. An in crease in the amount of added so dium hypochlorite causes a de crease
in the con cen tra tion of denatonium ben zo ate. In tro duc tion of so dium hypochlorite at a con cen tra tion of 12–15% ac tive chlo rine
in an amount cor re spond ing to 0.05 ml per 1 litre of spirit de na tured by Bitrex at 3 mg/l caused a de crease in denatonium ions con -
cen tra tion in the so lu tion, whereas ad di tion of an amount cor re spond ing to 0.20 ml/l of spirit caused its dis ap pear ance. The lack of
denatonium ions in the so lu tion meant that the con cen tra tion of Bitrex in the so lu tion was be low the limit of de tec tion and, thus, it
was prac ti cally re moved. Such a small ad di tion of so dium hypochlorite does not sig nif i cantly in flu ence the pH of the so lu tion.
Dis ap pear ance of denatonium ions also caused a dis ap pear ance of the char ac ter is tic bit ter taste of Bitrex, which was as sessed by
means of an organoleptic ex am i na tion. Ad di tion of the men tioned amounts of so dium hypochlorite did not cause a de crease in the
con cen tra tion of ben zo ate ions. Since con sumer (bev er age) spir its do not con tain ben zo ate ions, their de tec tion in a seized al co -
holic bev er age in di cates with high prob a bil ity that it was pre vi ously de na tured by Bitrex. The ben zo ate ions can also be re moved
from so lu tion by ad di tion of so dium hypochlorite, but the amount re quired in this case is sig nif i cantly higher: ap prox i mately
80 ml per 1 litre of spirit de na tured by 3 mg of Bitrex. In this case, the pH of the so lu tion is sig nif i cantly raised (pH = 11–12),
which makes it un suit able for con sump tion. Such a prod uct would have to be fur ther pu ri fied be fore be ing in tro duced onto the il -
le gal mar ket.
Key words
De na tured al co hol; Denatonium ben zo ate; Re moval.
Re ceived 9 De cem ber 2005; ac cepted 30 De cem ber 2005
© by the Institute
of Forensic Research
ISSN 1230-7483
in per cep tion of its bit ter taste have also been ob served
in chil dren, adults and el derly per sons [10]. The data
in di cate that the ad di tion of denatonium ben zo ate
would be ex pected to sig nif i cantly re duce, but most
likely not elim i nate, the prob a bil ity of an ac ci den tal in -
ges tion of prod ucts pre pared on the ba sis of al co hol
de na tured by this sub stance [1, 6]. Wide spread ap pli -
ca tion of this com pound has started in the United
States. At the be gin ning of the nine ties, in Or e gon,
man da tory ad di tion of this denaturant to au to mo tive
prod ucts con tain ing at least 10% of eth yl ene gly col or
at least 4% of meth a nol was in tro duced [4]. In 1995,
the Toxic House hold Prod ucts Stat ute re quired the ad -
di tion of denatonium ben zo ate at a con cen tra tion of
30–50 ppm with the in ten tion of re duc ing the fre -
quency of se ri ous pae di at ric in tox i ca tions with these
prod ucts [9]. Since that time, many pa pers in di cat ing
the pre ven ta tive ac tion of denatonium ben zo ate have
been pub lished [2, 8, 11].
Denatonium ben zo ate is a de na tur ing agent for
ethyl al co hol, listed in the de cree of the Min is try of
Ag ri cul ture and Ru ral De vel op ment of 11 Au gust
2003 on agents per mit ted to de na ture ethyl al co hol [4].
Al though this com pound has been listed as a de na tur -
ing agent for many years, it is only in the last few years
that a sig nif i cant in crease in its use has been ob served.
There may be sev eral rea sons for this. Its ad di tion does
not in flu ence the ba sic physicochemical prop er ties of
ethyl al co hol, be cause the amount of denatonium ben -
zo ate that should be added in ac cor dance with the
above men tioned de cree is small, i.e. 0.3 g per 100 l of
100% ethyl al co hol. Bitrex can also be added in the
form of 20% al co holic so lu tion.
The only fea ture of ethyl al co hol that changes sig -
nif i cantly af ter ad di tion of Bitrex is its taste. Pre vi -
ously, the most pop u lar denaturant in Po land was
meth a nol. Un for tu nately, it led to many deaths of per -
sons con sum ing in dus trial al co hol. In 2002, meth a nol
was with drawn from the list of denaturants of ethyl al -
co hol. The sec ond im por tant rea son for the growth in
the pop u lar ity of Bitrex ap pli ca tion is the tax reg u la -
tions cur rently in force in Po land. Ac cord ing to the de -
cree of the Min is try of Fi nance dated 26 April 2004 on
ex emp tion from ex cise tax [5], “the sale of ethyl al co -
hol de na tured by denatonium ben zo ate in amounts not
less than 1 g per 100 dm3 of 100% vol. ethyl al co hol,
dyed vi o let by ad di tion of pure crys tal vi o let in an
amount not less than 160 mg per 100 dm3 of 100% vol.
ethyl al co hol, in in di vid ual con tain ers of vol ume up to
1 litre” is ex cluded from spe cial tax su per vi sion. It is
highly prob a ble that this clause led to great amounts of
ethyl al co hol de na tured by Bitrex be ing pres ent on the
il le gal mar ket. The in tense bit ter taste of al co hol de na -
tured by Bitrex, how ever, re mains a prob lem for il le gal
pro duc ers of al co holic bev er ages. How ever, pre vi ous
stud ies by the au thors [12] and op er a tional ma te ri als of
the po lice have in di cated that il le gal pro duc ers re move
Bitrex by ad di tion of so dium hypochlorite to the de na -
tured al co hol.
2. Ma te ri als and meth ods
The stan dard of denatonium ben zo ate pur chased
from Sigma-Aldrich was used in the study. Stan dard
so lu tions of Bitrex were pre pared by weigh ing out an
ap pro pri ate amount of denatonium ben zo ate and dis -
solv ing in rec ti fied spirit pro duced by Polmos War -
szawa, Po land. The ac cu racy of the Bitrex con centra-
tion in the pre pared so lu tion was as sessed in inter-lab -
o ra tory ex am i na tions. So dium hypochlorite at a con -
cen tra tion of 12–14% ac tive chlo rine, pur chased from
Idalia, was ap plied in the study.
De ter mi na tions were also per formed on ev i den tial
ma te rial. Sam ples of ethyl al co hol se cured over the
course of sev eral months in 2005 from a plant deal ing
with its pro cess ing were used for this pur pose. This
plant pur chased the de na tured al co hol and pro duced
dif fer ent prod ucts, in clud ing dis in fec tants, win dow
clean ers or wind screen washer fluid.
In or der to de ter mine the denatonium ben zo ate
con tent of the ex am ined liq uids, high-per for mance liq -
uid chro ma tog ra phy (HPLC) was ap plied. Mea sure -
ments were per formed us ing a LaChrom (Merck/Hi -
tachi) in stru ment, which was equipped with a di ode-
ar ray de tec tor (DAD). Chro mato graphic sep a ra tion
was achieved on a Chromolith Per for mance RP-18e
(100 ´ 4.6 mm) mono lithic col umn. Mea sure ments of
denatonium ben zo ate con cen tra tion were taken at two
wave lengths: 205 nm and 230 nm. Mass spec trom e try
(MS) was ap plied to con firm the pres ence of dena-
tonium ben zo ate. A Quattro Mi cro API mass spec -
trom e ter man u fac tured by Micromass was ap plied in
the study. Both pos i tive (ES+) and neg a tive (ES–) ion -
is ation mode were used. The an a lyt i cal de tails of both
chro mato graphic meth ods have been pub lished in an -
other pa per by the au thors [12].
In or der to as sess the in flu ence of so dium
hypochlorite on the con tent of denatonium ben zo ate in
de na tured al co hol, a stock stan dard so lu tion was pre -
pared by ad di tion of Bitrex to rec ti fied al co hol. The
min i mal amount re quired by the de cree of the Min is try
of Ag ri cul ture and Ru ral De vel op ment, that is 3 mg/l,
was used. Then, 100-ml por tions of so lu tion were mea -
sured into flasks and so dium hypochlorite at a con cen -
tra tion of 12–14% ac tive chlo rine was added in the
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
Assessment of the efectiveness of denatonium benzoate (Bitrex)... 289
fol low ing amounts: 0 µl, 5 µl, 10 µl, 15 µl and 20 µl. In
or der to main tain a con stant to tal vol ume of the so lu -
tion, ap pro pri ate vol umes were re moved from the
flasks be fore ad di tion of hypochlorite. This en sured
that the con cen tra tion of Bitrex did not de crease due to
sam ple di lu tion. An iden ti cal pro ce dure was re peated
with greater ad di tions of so dium hypochlorite. 10-ml
por tions of de na tured al co hol were mea sured into
flasks and so dium hypochlorite was added in the fol -
low ing amounts: 0 µl, 50 µl, 100 µl, 200 µl, 400 µl,
600 µl and 800 µl. The above amounts were se lected
on the ba sis of pre lim i nary stud ies.
3. Re sults and dis cus sion
The HPLC method used by the au thors [12] al -
lowed two chro mato graphic peaks to be ob tained dur -
ing anal y sis of stan dard so lu tions of denatonium
ben zo ate. The first peak at re ten tion time 9.05 min cor -
re sponded to ben zo ate ions, whereas the sec ond one, at
re ten tion time 10.30 min, was de pend ent on the con -
cen tra tion of denatonium ions (it orig i nated from
denatonium ions or from undissociated mol e cules of
denatonium ben zo ate). Mea sure ment was per formed
at 230 nm for the ben zo ate ion and at 205 nm for the
ion de pend ent on denatonium con cen tra tion.
The ad di tion of so dium hypochlorite to ethyl al co -
hol de na tured by Bitrex causes for ma tion of a white
ge lat i nous pre cip i ta tion. Its vol ume and the re sults of
el e men tal anal y sis per formed by means of the
SEM-EDX method as well as spec tro met ric anal y sis in
the in fra-red re gion (FT-IR) in di cate that the main
com po nent is so dium hypochlorite. The pre cip i ta tion
of so dium hypochlorite af ter ad di tion to de na tured al -
co hol could re sult from the lower sol u bil ity of this salt
in al co hol com pared with wa ter.
In or der to re move the formed pre cip i tates, the
sam ples were cen tri fuged. Then, the liq uid lo cated
above the pre cip i tate was trans ferred into mea sure -
ment vi als and HPLC anal y sis was per formed. In or der
to as sess the mea sure ments pre ci sion, three sam ples
were col lected from each so lu tion. The re sults are
shown in Fig ure 1.
As can be seen from data shown in Fig ure 1, the ad -
di tion of so dium hypochlorite sig nif i cantly in flu ences
the con tent of denatonium ben zo ate in de na tured
spirit. Add ing as lit tle as ap prox i mately 5 µl per
100 ml of spirit, which is equiv a lent to ap prox i mately
0.05 ml per 1 litre of spirit, causes a de crease in peak
area at re ten tion time 10.30 min, i.e. denatonium ions
or undissociated mol e cules of denatonium ben zo ate
(whose con cen tra tion also de pends on the con cen tra -
tion of denatonium ions). This means that the con cen -
tra tion of denatonium ben zo ate has been low ered and,
there fore, the ac tion of the denaturant has been weak -
ened. Ac cord ing to Pol ish law, such ac tiv ity is a crime.
In ac cor dance with Ar ti cles 13 and 14 of the Law dated
2 March 2001 on spirit pro duc tion, pro duc tion and
bot tling of spirit prod ucts and pro duc tion of to bacco
prod ucts [3] “any per son that pu ri fies de na tured spirit
or oth er wise re duces the ac tion of a de na tur ing agent is
li a ble to a fine, a pen alty of re stric tion of lib erty, or im -
pris on ment of up to 1 year. If the crime was the reg u lar
source of in come for the per pe tra tor, the per son is li a -
ble to a pen alty of im pris on ment up to 3 years”.
Add ing 20 µl of so dium hypochlorite to 100 ml of
de na tured al co hol caused a dis ap pear ance of the peak
at re ten tion time 10.30 min. This means that the con -
cen tra tion of Bitrex in so lu tion was be low the limit of
de tec tion, which is 0.1 mg/l. Such a small ad di tion of
so dium hypochlorite does not sig nif i cantly in flu ence
the pH of the so lu tion. The re sult of per formed
organoleptic ex am i na tions of the above liq uid was
con sis tent with ex pec ta tions, in di cat ing that the char -
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
290 D. Zuba, C. Œwiegoda, B. Byrska et al.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.00 0.05 0.10 0. 15 0.20
Volume of sodium hypochlorite adde d to 1 litre of denature d spirit [ml ]
Concentration[mg/l]
Denatonium
Benzoate
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
020 40 60 80 100
Volume of sodium hypochlorite adde d to 1 litre of denature d spirit [ml]
Concentration[mg/l]
Denatonium
Benzoate
Fig. 1. The re la tion ship be tween the ar eas of chro mato -
graphic peaks orig i nat ing from denatonium ben zo ate and the
amounts of so dium hypochlorite added to de na tured spirit.
ac ter is tic bit ter taste of Bitrex had dis ap peared. The
ob tained prod uct was dif fi cult to dis tin guish from con -
sumer (bev er age) al co hol.
Fur ther ad di tion of so dium hypochlorite to al co hol
de na tured by Bitrex can also lead to the dis ap pear ance
of the peak at re ten tion time 9.05 min, i.e. ben zo ate
ions. But the amount of so dium hypochlorite re quired
for this pur pose is very large and amounts to about
80 ml per 1 litre of de na tured spirit. Such great ad di -
tion of a salt of a weak acid and strong base causes
a sig nif i cant rise in pH (pH = 11–12). The con sump -
tion of a strongly ba sic liq uid is a threat to the health or
even life of con sum ers. In pass ing, it should be noted
that in the pre vi ous year, spir its with a very strong ba -
sic chem i cal re ac tion were de liv ered for in ves ti ga tion
to the In sti tute of Fo ren sic Re search. This may con -
firm the the sis that the men tioned pro ce dure is ap plied
by il le gal pro duc ers of al co holic bev er ages. On the
other hand, it seems that the ba sic pH of spirit from
which Bitrex has been re moved can be sim ply elim i -
nated, for ex am ple, by add ing an ap pro pri ate amount
of acid.
The above re sults in cline us to wards the con clu sion
that af ter ad di tion of so dium hypochlorite to al co hol
de na tured by Bitrex, there is a re ac tion of hypochlorite
an ions with denatonium cat ions and prob a bly co-pre -
cip i ta tion of them with so dium hypochlorite en sues.
The added so dium hypochlorite can also ox i dize the
denatonium ions (hypochlorite is a strong ox i dant,
whereas Bitrex is sen si tive to such sub stances). The
oc cur ring pro cesses cause a de cline in Bitrex con cen -
tra tion in so lu tion. Add ing large amounts of so dium
hypochlorite also causes the dis ap pear ance of the sec -
ond ion orig i nat ing from Bitrex. This can re sult from
the pre cip i ta tion of so dium ben zo ate as well as from
ox i da tion of com pounds pres ent in so lu tion.
The above re sults were con firmed by means of
mass spec trom e try. Dur ing anal y sis of al co hol de na -
tured by Bitrex in pos i tive-ion is ation mode, the peak
cor re spond ing to the mass of the denatonium ion
(m/z = 325) was ob tained, whereas in neg a tive-ion is -
ation mode – the peak cor re spond ing to the mass of
ben zo ate ion (m/z = 121). Next, sam ples of al co hol de -
na tured by Bitrex with a small ad di tion of so dium
hypochlorite (10 µl per 100 ml of de na tured al co hol)
were ana lysed. In pos i tive-ion is ation mode, be side the
ion at m/z ra tio = 325, an ad di tional peak of mass 360
ap peared (mass greater than that of cho rine). In neg a -
tive-ion is ation mode, the con fig u ra tion of peaks char -
ac ter is tic for chlo ride de riv a tives was ob tained.
Anal y sis of de na tured spirit, to which was added so -
dium hypochlorite in an amount caus ing dis ap pear -
ance of the peak at re ten tion time 10.30 min on the
HPLC chromatogram, in di cated dis ap pear ance of the
peak at m/z = 325, cor re spond ing to denatonium ions.
Anal y sis of the so lu tion in neg a tive-ion is ation mode
con firmed the pres ence of chlo ride de riv a tives. The
mass spec tra of the men tioned so lu tions in pos i tive-
ion is ation mode are shown in Fig ure 2, whereas in
neg a tive-ion is ation mode – in Fig ure 3.
4. Re sults of ev i den tial sam ples anal y ses
The worked out method of denatonium ben zo ate
de ter mi na tion and the re sults of the per formed ex per i -
ments were uti lised in ex pert opin ions. The re sults of
ex am i na tions ob tained in one of the cases are pre -
sented be low.
A proc u ra tor con duct ing an in ves ti ga tion sus pected
that, in a plant deal ing with pro cess ing of de na tured al -
co hol, the denaturant, i.e. Bitrex, was be ing re moved.
The mech a nism of this pro cess was not known. The
plant bought the de na tured al co hol and it was used for
pro duc tion of dif fer ent prod ucts, e.g. an ti bac te rial fluid,
win dow clean ers or wind screen washer fluid. The liq -
uids and cop ies of plant norms used in pre par ing of the
prod ucts were sub mit ted for ex am i na tion.
Re sults of anal y ses by means of high-per for mance
liq uid chro ma tog ra phy are shown in Ta ble I.
TA BLE I. THE CON CEN TRA TION OF DENATONIUM
BEN ZO ATE CAL CU LATED BY THE IN DI RECT
METHOD US ING THE CON CEN TRA TION OF
BEN ZO ATE IONS (Cben zo ate) AND DENATONIUM
IONS OR UNDISSOCIATED MOL E CULES OF
DENATONIUM BEN ZO ATE (Cdenatonium)
Ev i dence nr Cbenzoate [mg/l] Cdenatonium [mg/l]
11.2 1.0
21.3 1.1
31.1 –
41.1 –
51.9 –
61.8 –
71.4 –
81.8 –
91.4 –
10 1.7 –
11 2.1 –
12 11.5 9.1
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
Assessment of the efectiveness of denatonium benzoate (Bitrex)... 291
As can be seen from data in Ta ble I, the ev i den tial
spir its can be di vided into two or three groups. In ev i -
dences 1 and 2, the con cen tra tions of denatonium ben -
zo ate cal cu lated on the ba sis of two dif fer ent
chro mato graphic peaks were equal (within the range
of an a lyt i cal er ror of the method). In ev i dences 3–11,
the pres ence of ben zo ate ions was as cer tained, but the
sec ond peak at re ten tion time 10.30 min was not de -
tected on HPLC chromatograms. In the case of the liq -
uid from ev i dence 12, the con cen tra tion of Bitrex
cal cu lated on the ba sis of ben zo ate ions con tent was
higher than that cal cu lated on the ba sis of anal y sis of
the sec ond ion and the dif fer ence exceeded the
analytical error significantly.
The above liq uids were also sub jected to anal y sis
by means of mass spec trom e try. In liq uids of ev i -
dences 1 and 2, the pres ence of denatonium ions was
con firmed (and thus that of Bitrex). Fur ther more, the
re sults of the stud ies in di cated that the above men -
tioned liq uids do not con tain prod ucts of the re ac tion
be tween denatonium ben zo ate and so dium hypo chlo -
rite. The con cen tra tions of Bitrex de ter mined in the
liq uids of ev i dences 1 and 2 were lower than the
amount re quired ac cord ing to the de cree of the Min is -
try of Ag ri cul ture and Ru ral De vel op ment and there -
fore, ac cord ing to the reg u la tion be ing in force, could
not be clas si fied as de na tured ethyl al co hol. On the
other hand, it has been ob served in the prac tice of the
In sti tute of Fo ren sic Re search that 20% tech ni cal so lu -
tion of Bitrex con tains sig nif i cantly lower amounts of
this sub stance. The con cen tra tion of Bitrex de ter mined
in such liq uids by means of the HPLC method was
10–12%. The liq uids used for de na tur ation are ex am -
ined be fore their use, but the an a lyt i cal meth ods ap -
plied for this pur pose are highly non-se lec tive
(mea sure ment of den sity is used most of ten). Thus, it is
pos si ble that the lower con tent of Bitrex de ter mined in
the liq uids of ev i dences 1 and 2 re sulted from its lower
con tent in the de na tur ing fluid.
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
292 D. Zuba, C. Œwiegoda, B. Byrska et al.
m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
%
0
100
Scan ES+
2.54e6
68.64
63.64
62.21
325.02
80.72 134.76
90.63 114.71 128.68
143.79
170.83 202.81
174.72 246.83
212.89 238.79 255.91 304.74
278.13 300.88
326.03
347.09 383.10 397.04
m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
%
0
100
Scan ES+
4.63e6
68.54
63.54
62.08
80.54
70.10
82.61 324.98
134.77
114.78
90.61 138.65 170.72 192.76 198.78 276.94
250.74
215.83 270.84 298.90 304.59
359.95
327.00 380.94 385.04
m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
%
0
100
Scan ES+
1.83e6
68.60
63.66
62.17
80.54
70.36 128.69
82.61 114.85
109.83 170.84
138.65 152.97 186.67 380.94
352.77
234.70
196.60
216.77
244.57 294.56
256.63 284.61 304.63 332.42 362.54 397.02
Fig. 2. Mass spec tra of al co hol de na tured by Bitrex in pos i tive-ion is ation mode: a) with out so dium hypochlorite, b) af ter ad di tion
of so dium hypochlorite in the amount of 100 µl/l, c) af ter ad di tion of so dium hypochlorite in the amount of 80 ml/l.
Spec tro met ric anal y sis of the liq uids of ev i dences
3–11 con firmed that they did not con tain denatonium
ions (and there fore they did not con tain Bitrex),
whereas the pres ence of mass peaks that could orig i -
nate from the prod ucts of re ac tion be tween dena to -
nium ben zo ate and so dium hypochlorite were detec-
ted. There fore, one can as sume that liq uids of ev i -
dences 3–11 con tained denatonium ben zo ate (Bitrex)
and it was re moved af ter wards, e.g. by ad di tion of so -
dium hypochlorite.
Anal y sis of the liq uid of ev i dence 12 by means of
mass spec trom e try con firmed that it con tains dena -
tonium ben zo ate (Bitrex). In the ob tained mass spec -
tra, both in pos i tive- and neg a tive-ion is ation mode,
ad di tional peaks that prob a bly came from prod ucts of
the re ac tion be tween denatonium ben zo ate and so dium
hypochlorite were ob served. They were iden ti cal to
those ob tained dur ing the anal y sis of stan dard so lu -
tions of denatonium ben zo ate in spirit to which so dium
hypochlorite had been added. This con firmed the hy -
poth e sis re sult ing from anal y sis by means of the HPLC
method that some of the denatonium ions were re -
moved from ev i dence liq uid 12, prob a bly by ad di tion
of so dium hypochlorite.
5. Con clu sions
The re sults of per formed stud ies show un am big u -
ously that the ad di tion of so dium hypochlorite to ethyl
al co hol de na tured by Bitrex causes par tial or to tal re -
moval of this denaturant. The ef fec tive ness of re moval
de pends on the vol ume of so dium hypochlorite added
to the so lu tion.
The worked out pro ce dure of spirit prod ucts ex am -
i na tion by means of the HPLC method and mass spec -
trom e try al lows de ter mi na tion of the con cen tra tion of
denatonium ben zo ate (Bitrex) by the de ter mi na tion of
ben zo ate ions con tent as well as the con tent of ben zo -
ate ions or mol e cules of denatonium ben zo ate. Anal y -
sis of the con tent of these com po nents al lows us to
es tab lish whether the ex am ined liq uid con tains Bitrex,
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
Assessment of the efectiveness of denatonium benzoate (Bitrex)... 293
m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100
Scan ES-
1.17e588.69
76.75
59.82
76.19
120.33
90.65
156.65
150.82 407.12
188.91 238.65
224.79 324.14
259.06 282.80 302.65 397.09
352.95 380.38 491.32
445.26 481.04
m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
%
0
100
Scan ES-
1.17e5
82.54
76.79
59.90
84.61
92.55
94.62 188.55
140.60
102.63
118.58
150.45 186.93
167.83
190.63
326.37
256.85200.74 208.62 222.60 247.89 266.54 276.67 314.99 358.19
348.49 396.89
382.46
378.93
m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
%
0
100
Scan ES-
7.20e4
82.44
80.64
70.06
188.62
140.6684.49
92.71 100.92 116.47
142.61
154.83
180.64
190.56
268.69
192.57
248.98
206.53218.72 354.54
314.61
296.38
270.76 318.60 346.78 384.53
362.52 388.57
Fig. 3. Mass spec tra of al co hol de na tured by Bitrex in neg a tive-ion is ation mode: a) with out so dium hypochlorite, b) af ter ad di tion
of so dium hypochlorite in the amount of 100 µl/l, c) af ter ad di tion of so dium hypochlorite in the amount of 80 ml/l.
or whether it was de na tured by Bitrex and pu ri fied af -
ter wards. This in for ma tion is of great im por tance in
the prep a ra tion of ex pert opin ions for courts.
Ref er ences:
1. Berning C. K., Grif fith J. F., Wild J. E., Re search on the
ef fec tive ness of denatonium ben zo ate as a de ter rent to liq -
uid de ter gent in ges tion by chil dren, Fun da men tals of Ap -
plied Tox i col ogy 1982, 2, 44–48.
2. Carnahan R. M., Kutscher E. C., Obritsch M. D. [et al.],
Acute eth a nol in tox i ca tion af ter con sump tion of hair -
spray, Pharmacotherapy 2005, 25, 1646–1650.
3. Dziennik Ustaw z dnia 11 kwietnia 2001 r., nr 31, poz.
353.
4. Dziennik Ustaw z dnia 11 sierpnia 2003 r., nr 163, poz.
1582.
5. Dziennik Ustaw z dnia 1 maja 2004 r., nr 97, poz. 966.
6. Hansen S. R., Janssen C., Beasley V. R., Denatonium ben -
zo ate as a de ter rent to in ges tion of toxic sub stances: tox ic -
ity and ef fi cacy, Vet er i nary and Hu man Tox i col ogy 1993,
35, 234–236.
7. Henderson M. C., Neumann C. M., Buhler D. R., Anal y sis
of denatonium ben zo ate in Or e gon con sumer prod ucts by
HPLC, Chemosphere 1998, 36, 203–210.
8. Jack son M. H., Payne H. A., Bittering agents: their po ten -
tial ap pli ca tion in re duc ing in ges tions of en gine cool ants
and wind shield wash, Vet er i nary and Hu man Tox i col ogy
1995, 37, 323–326.
9. Mul lins M. E., Zane Horowitz B., Was it nec es sary to add
Bitrex (denatonium ben zo ate) to au to mo tive prod ucts?,
Vet er i nary and Hu man Tox i col ogy 2004, 46, 150–152.
10. Schiffman S. S., Gatlin L. A., Frey A. E., [et al.], Taste
per cep tion of bit ter com pounds in young and el derly per -
sons: re la tion to lipophilicity of bit ter com pounds, Neuro -
biology of Ag ing 1994, 15, 743–750.
11. Sibert J. R., Frude N., Bittering agents in the pre ven tion of
ac ci den tal poi son ing: chil dren’s re ac tions to denatonium
ben zo ate (Bitrex), Ar chives of Emer gency Med i cine 1991,
8, 1–7.
12. Zuba D., Œwiegoda C., Byrska B. [et al.], De ter mi na tion
of denatonium ben zo ate (Bitrex) in denaturated spir it
preparations, Prob lems of Fo ren sic Sci ences 2005, 63,
275–287.
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
294 D. Zuba, C. Œwiegoda, B. Byrska et al.
Cor re spond ing au thor
Dariusz Zuba
Instytut Ekspertyz S¹dowych
ul. Westerplatte 9
31-033 Kraków
e-mail: dzuba@ies.krakow.pl
1. Wpro wad zenie
Ben zoe san de nat oni um (Bi trex) jest sub stancj¹ sto -
sow an¹ od wie lu lat na ca³ym œwi ecie do ska ¿ania alko -
ho lu prze znac zone go do celów prze mys³owych. G³ówny
cz³on na zwy tego zwi¹zku, de nat oni um, zwi¹zany jest
z tym za stos owa niem, po niew a¿ po chod zi od angiel -
skiego s³owa „de nat ura ted”, czy li ska ¿ony (st¹d te¿ po -
chod zi pol ska na zwa „de nat urat”). Cha rakt ery zuje siê on
bar dzo in tens ywnym, gorz kim sma kiem, co po wod uje, ¿e
ju¿ nie wielki do dat ek Bi trexu czy ni wyr ób nie nad aj¹cym
siê do spo ¿yc ia. Z tego wzg lêdu jest idea lnym do datk iem
za pob iegaj¹cym przy padk owe mu przyj mow aniu przez
lu dzi ta kich p³ynów, jak de terg enty, œr odki de zynf eku -
j¹ce, p³yny czyszcz¹ce, œrodki wy kor zyst ywane w og ród -
kach do mow ych czy pro dukty ogrodn icze. W ba dan iach
za uwa ¿ono ró ¿ni ce w per cepc ji jego gorz kiego sma ku
u dzie ci, do ros³ych i osób star szych [10]. Dane wska zuj¹,
¿e do dat ek ben zoe sanu de nat oni um zda je siê w znacz¹cy
sposób zmniejszaæ, choæ nie do koñca eliminowaæ, praw -
dopodobieñstwo przy padk owe go spo¿ ycia prod uktów
spo rz¹dzo nych na ba zie spi ryt usu ska ¿on ego tym zwi¹z -
kiem [1, 6]. Po wszechn ie za czêto st osowaæ ten zwi¹zek
w Sta nach Zjed noc zony ch, gdzie na pocz¹tku lat dzie -
wiêædziesi¹tych w sta nie Oreg on wpro wad zono koni ecz -
noœæ do daw ania go do pr odu któw sto sow any ch w samo -
cho dach, a za wier aj¹cych co naj mniej 10% gli kolu etyl e -
no wego lub co naj mniej 4% me tan olu [7]. Wy dane
w 1995 roku roz porz¹dze nie do tycz¹ce to ksycznoœci pro -
duktów gospo darstwa do mow ego uznawa³o wy móg do -
daw ania do nich ben zoe sanu de na ton ium w st ê¿e niu
30–50 ppm w celu zmniej szen ia czêsto œci powa¿n ych
zatruæ dzie ci tymi pro dukt ami [9]. Od tego cza su opub -
liko wano wie le prac wy kaz uj¹cych na jego pre wenc yjne
dzia³anie [2, 8, 11].
Ben zoe san de nat oni um jest œr odk iem sk a¿aj¹cym al -
koh ol etyl owy zgod nie z roz porz¹dze niem Mi nis tra Rol -
nict wa i Roz woju Wsi z dnia 11 sierp nia 2003 r.
w spra wie œrod ków do puszc zony ch do sk a¿ania al koh olu
etyl owe go [4]. Wpraw dzie zwi¹zek ten znaj dowa³ siê na
wy kaz ie œr odk ów sk a¿aj¹cych ju¿ od wie lu lat, to do piero
w ostatn ich la tach za uwa¿ono znacz ny wzrost jego u¿y -
cia. Wynikaæ to mo¿e z kil ku przy czyn. Jego do dat ek nie
wp³ywa na pod staw owe w³asn oœci fi zyk och emic zne al -
koh olu etyl owe go, poni ewa¿ iloœæ ben zoe sanu denato -
nium, jak¹ nal e¿y dod aæ zgod nie z ww. roz po rz¹dze-
niem, jest nie wielka i wy nosi 0,3 g na 100 l 100% al koh o -
lu etyl owe go. Bi trex mo¿e byæ do dany ró w ni e¿ w po staci
20% roz tworu al koh olo wego. Je dyn¹ ce ch¹ al koh olu
ety l owe go, któr¹ w znacz¹cy sposób zmie nia do dat ek Bi -
trexu, jest jego smak. Po przedn io naj pop ula rnie jszym
ska¿alnikiem w Pol sce by³ me tan ol, co nie stety do prow a -
dzi³o do wie lu zgo nów osób spo ¿y wa j¹cych al koh ol
prze mys³owy. Me tan ol zo sta³ w 2002 ro ku wy cof any
z lis ty sub stanc ji sto sow any ch jako ska ¿al niki al koh olu
etyl owe go. Dru gim istotn ym czynni kiem od pow iedz ial -
nym za wzrost popularnoœci sto sow a nia Bi trexu s¹ obo -
wi¹zuj¹ce obecn ie prze pisy po datk owe. Zgod nie z roz -
porz¹dze niem Mi nis tra Fi nan sów z dnia 26 kwiet nia
2004 r. w spra wie zwol nieñ od po datku ak cyz owe go [5],
ze sz cze gól nego nad zoru podat ko wego wy³¹cza siê
„ sprze da¿ al koh olu etyl owe go ska¿one go ben zoe sanem
de nat oni um w il oœci nie mniej szej ni¿ 1 g/100 dm3 alko -
ho lu etyl owe go 100% vol., zabar wionego na fio let owo
przez do dan ie czy stego fio letu kry stal iczne go w ilo œci nie
mniej szej ni¿ 160 mg/100 dm3 al koh olu etyl owe go
100% vol., w opak owa nia ch jed nostk owy ch o poj emnoœ -
ci do 1 li tra”. Za pis ten spo wod owa³ praw dop odo bnie, ¿e
du¿a iloœæ al koh olu etyl owe go ska¿ one go Bi trexem zna -
laz³a siê na nie leg alnym ryn ku. Pro blem em dla niele -
galnych pro duc entów nap ojów al koh olo wych po zos ta-
wa³ jed nak in tens ywny, gorz ki smak al koh olu sk a¿o nego
Bi trexem. Jed nak, jak wy nika³o z po przedn ich ba dañ
[12] oraz ma ter ia³ów oper acy jnych po lic ji, nie leg alni
pro duc enci usuw aj¹ Bi trex po przez do dan ie do sk a¿o -
nego al koh olu pod chlor ynu sodu.
2. Ma ter ia³ i me tody
W ba dan iach wy kor zyst ano wzo rzec ben zoe sanu de -
nat oni um za kup iony w fir mie Sig ma-Al dr ich. Roz twory
wzor cowe Bi trexu sporz¹dza no po przez od wa¿enie od -
pow iedni ej il oœci ben zoe sanu de nat oni um i ro zcieñc ze -
nie go spi ryt usem rek tyf iko wan ym pro duk owa nym przez
Pol mos War szawa. Dok³adn oœæ stê ¿enia Bi trexu w spo -
rz¹dzo nym roz twor ze zo sta³a ocen iona w ra mach ba dañ
mi êdzylaboratoryjnych. Do ba dañ za stos owa no ró wnie¿
pod chlor yn sodu fir my Idal ia o stê¿ eniu 12–14% aktyw -
nego chlo ru.
Po nadto prze prow adz ono oznac zenia w ma ter iale do -
wod owym. Wy kor zyst ano w tym celu pr óbki al koh olu
etyl o we go za bezp iecz onego w prze ci¹gu kil ku mie siêcy
2005 ro ku w zak³adzie zaj muj¹cym siê jego prze twór -
stwem. Zak³ad ten ku powa³ sk a¿o ny al koh ol, z kt óre go
wy rab ia³ ró¿ne pro dukty, np. p³yny an tyb akte ryj ne,
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
OCENA EFEKTYWNOŒCI METODY USUWANIA BENZOESANU
DENATONIUM (BITREXU) ZE SKA¯ONYCH WYROBÓW
SPIRYTUSOWYCH ZA POMOC¥ PODCHLORYNU SODU
p³yny do my cia szyb czy p³yny do spry skiw ania szyb sa -
moc hodo wych.
W celu wy znac zenia za wart oœci ben zoe sanu de nat o -
ni um w ba dan ych p³ynach za stos owa no me todê wyso ko -
sprawnej chro mat ogr afii cie czow ej (HPLC). Ba dan ia
prze prow adz ono za po moc¹ urz¹dze nia La Chrom
(Merck/Hitac hi) wy pos a¿onego w de tekt or sze regu diod
(DAD). Roz dzia³ chro mat ogr afic zny by³ pro wad zony na
ko lumn ie mo nol ity cznej Chro mol ith Per form ance RP-18e
(100 ´ 4,6 mm). Po miary st ê¿en ia ben zoe sanu de nat o ni um
by³y wy kon ywa ne przy dw óch d³ug oœc iach fali: 205 nm
i 230 nm. Do po twierd zenia ob ecnoœci ben zoe sanu dena -
tonium wy kor zyst ano me todê spek trom etr ii ma sow ej
(MS). W ba dan iach za stos owa no spek trom etr ma sowy
Qu att ro Micro API fir my Micro mass. Pomia rów do kon y -
wa no za rów no przy do datn iej, jak i ujemn ej joni za cji.
Szcze gó³y anal ity czne obu me tod chro mat ogr afic z nych
zo sta³y opub liko wane w odrê bnej pra cy aut orów [12].
W celu oceny wp³ywu pod chlor ynu sodu na zaw ar -
toœæ ben zoe sanu de nat oni um w sk a¿o nym al koh olu spo -
rz¹dzo no pod staw owy roz twór wzor cowy po przez do -
da nie do spi ryt usu rek tyf iko wan ego Bi trexu w mini -
malnej il oœci wy mag anej przez roz porz¹dze nie Mi nis tra
Rol nict wa i Roz woju Wsi, tj. 3 mg/l. Nas têp nie do kolb
mia row ych od mier zono po 100 ml tego roz tworu i do -
dano pod chlor yn sodu o stê ¿eniu 12–14% ak tywn ego
chlo ru w nast êpuj¹cych iloœ ciach: 0 µl, 5 µl, 10 µl, 15 µl
i 20 µl. Przed do dan iem pod chlor ynu usu niêto z kolb od -
pow iednie ob jêt oœci roz tworu, aby nie zwi êksz aæ ca³ko -
wit ej ob jêt oœci roz tworu, a tym sa mym nie zm niejszaæ
st ê¿enia Bi trexu. Ident yczny eks per yme nt przeprowa -
dzono dla wiêk szych doda tków pod chlor ynu sodu. Do
kolb mia row ych od mier zono po 10 ml roz tworu ska ¿one -
go al koh olu i do dano pod chlor yn sodu w nas têpuj¹cych
ilo œciach: 0 µl, 50 µl, 100 µl, 200 µl, 400 µl, 600 µl
i 800 µl. Pow y¿sze ilo œci zo sta³y do brane na pod staw ie
bad añ wst êpny ch.
3. Wy niki i ich om ówi enie
Sto sow ana przez autorów me toda HPLC [12] po z -
woli³a na uzys kanie pod czas anal izy rozt wor ów wzor -
cow ych ben zoe sanu de nat oni um dw óch pi ków chro ma-
to graf iczny ch, z któr ych pierw szy, o cza sie re tenc ji
9,05 min, od pow iada³ jo nowi ben zoe sano wemu, nato -
miast dru gi, o cza sie re tenc ji 10,30 min, by³ za le¿ny od
stê ¿enia jo nów de nat oni um (po chod zi³ b¹dŸ to od sa -
mych jo nów b¹dŸ te¿ nie zdys ocj owan ych cz¹ste czek
ben zoe sanu de nat oni um). Dla jonu ben zoe sano wego po -
miar zo sta³ prze prow adz ony przy 230 nm, na tom iast dla
jonu za le¿ nego od st ê¿e nia de nat oni um – przy 205 nm.
Do dan ie pod chlor ynu sodu do al koh olu etyl owe go
ska¿ one go Bi trexem po wod uje wytr¹ce nie bia³ego, se -
row ate go osadu. Jego objê toœæ oraz wy niki anal izy pier -
wiastk owej prze prow adz onej za po moc¹ me tody
SEM-EDX i anal izy spek trom etr ycznej w pod czerw ieni
(FT-IR) wska zuj¹, ¿e jego g³ównym sk³ad nik iem jest
pod chlor yn sodu. Wytr¹ce nie siê pod chlor ynu sodu po
do dan iu do roz tworu ska ¿one go al koh olu mo¿e wy nik aæ
z mniej szej rozpu szczalnoœci tej soli w al koh olu w po -
rów nan iu z wod¹.
W celu us uni êcia po wsta³ych os adów pr óbki od wir o -
wa no. Na stê pnie p³yn znad osadu prze nies iono do na -
czyn iek po miar owy ch i pod dano anal izie me tod¹ HPLC.
W celu oceny pre cyz ji ozn acz eñ z ka¿ dego roz tworu po -
brano po trzy pr óbki. Wy niki przed staw iono na ry cin ie 1.
Jak wy nika z tych da nych, do dat ek pod chlor ynu sodu
wp³ywa istotn ie na zawart oœæ ben zoe sanu de nat oni um
w sk a¿o nym al koh olu. Do dan ie ju¿ nie wielk iej jego il oœ -
ci (rzê du 5 µl na 100 ml spi ryt usu, co od pow iada oko³o
0,05 ml na 1 litr spi ryt usu) po wod uje zmniej szen ie po -
wierzchni piku o cza sie re tenc ji 10,30 min, czy li jon ów
de nat oni um lub niezdysocjowa nych cz¹ste czek benzo -
esanu de nat oni um (ró wni e¿ za le¿ nych od st ê¿e nia jo nów
de nat oni um). Oznac za to spa dek stê ¿enia ben zoe sanu de -
nat oni um w sk a¿o nym spi ryt usie, a tym sa mym os³abie-
nie dzia³ania œrodka ska¿aj¹cego. Taka dzia³alnoœæ we -
d³ug pol skiego pra wa jest prze stêpstwem. Zgod nie z art.
13 i 14 ustawy z dnia 2 mar ca 2001 r. o wy rob ie spi ryt usu,
wy rob ie i roz lew ie wyr obów spi ryt uso wych oraz wy -
twar zaniu wy robów ty ton iowy ch [3] „kto od ka¿a spiry -
tus ska ¿ony lub w ja kik olwi ek sp osób os³abia dzia³anie
œr odka sk a¿aj¹cego, pod lega grzyw nie, ka rze ogran icz e -
nia wolno œci albo po zbaw ienia wo lnoœ ci do roku. Je ¿eli
z pope³nia nia tego prz estêpstwa spraw ca uczyni³ so bie
sta³e Ÿród³o do chodu, pod lega ka rze po zbaw ienia wol -
noœci do lat 3”.
Do dan ie 20 µl pod chlor ynu sodu do 100 ml ska -
¿onego spi ryt usu spo wod owa³o za nik piku o cza sie re -
tenc ji 10,30 min, co oznac za, ¿e stê¿e nie Bi trexu w roz -
two rze by³o pon i¿ej gra nicy wyk ryw alnoœci me tody wy -
nosz¹cej 0,1 mg/l. Tak nie wielki do dat ek pod chlor ynu
sodu nie wp³yn¹³ w istotny sposób na od czyn che miczny
roz tworu (pH). Prze prow adz one ba dan ia or gan ole ptyc z -
ne po wy¿ sze go p³ynu wska za³y zgod nie z oczek iwa niem,
¿e gorz ki smak cha rakt ery sty czny dla Bi trexu za nik³,
a otrzym any pro dukt by³ trud ny do odró ¿nienia od alko -
holu kon sumpc yjne go.
Dal sze do daw anie pod chlor ynu sodu do al koh olu ety -
l owe go ska¿ one go Bi trexem mo¿e do prowadziæ ró wnie¿
do za niku piku o cza sie re tenc ji 9,05 min, a wiêc jonu
ben zoe sano wego. Jed nak ilo œæ pod chlor ynu sodu, jak¹
nal e¿y u¿yæ w tym celu, jest bar dzo du¿a i wy nosi oko³o
80 ml na 1 litr sk a¿o nego spi ryt usu. Tak du¿y do dat ek
soli s³abego kwa su i moc nej za sady po wod uje znacz ny
wzrost od czynu che miczn ego (pH = 11–12). Spo ¿ycie
p³ynu o od czyn ie moc no za sad owym sta nowi jed nak za -
gr o¿e nie dla zdro wia, a na wet ¿y cia po tenc jalny ch kon -
sumentów. Na mar gin esie nale¿y zaz nac zyæ, ¿e w ubieg -
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
296 D. Zuba, C. Œwiegoda, B. Byrska i in.
³ym roku do In styt utu Eks pert yz S¹do wych do starc zono
do ba dan ia spi ryt usy maj¹ce sil nie al kal iczny od czyn. Po -
twierdzaæ to mo¿e tezê, ¿e pro ced er do daw ania pod -
chlo rynu sodu jest sto sow any przez nie leg alny ch prod u -
ce ntów nap ojów al koh olo wych. Wy daje siê jed nak, ¿e
od czyn za sad owy spi ryt usu, z któ rego usu niê to Bi trex,
mo ¿na w pro sty spo sób wye liminowaæ np. po przez do -
dan ie od pow iedni ej il oœci kwasu.
Po wy¿ sze wy niki sk³aniaj¹ do wnio sku, ¿e po doda -
niu pod chlor ynu sodu do al koh olu etyl owe go ska¿ one go
Bi trexem do chod zi do re akc ji an ion ów pod chlor yno -
wych z ka tion ami de nat oni um i na stê puje praw dop odo b -
nie ich wspó³str¹ca nie ze str¹ca niem sa mego pod chlo-
rynu sodu. Do dany pod chlor yn sodu mo¿e rów nie¿ utl e -
niaæ jony de nat oni um (pod chlor yn nal e¿y do sil nych
utlen iaczy, na które Bi trex jest wr a¿liwy). Za chodz¹ce
pro cesy po wod uj¹ spa dek stê ¿enia Bi trexu w roz twor ze.
Do dan ie du ¿ych il oœci pod chlor ynu sodu po wod uje za nik
dru giego jonu po chodz¹cego z Bi trexu. Mo¿e to wy nikaæ
z wytr¹ce nia ben zoe sanu sodu lub te¿ mo¿e doc hod ziæ do
utlen iania zwi¹zków obecn ych w roz twor ze.
Po wy¿s ze wy niki po twierd zono ba dan iami z za stos o -
wa niem spek trom etr ii mas. W trak cie anal izy spi ryt usu
ska ¿on ego Bi trexem w jo niz acji do datn iej uzys kano pik
od pow iadaj¹cy mas¹ jo nowi de nat oni um (m/z = 325), na -
tom iast w jo niz acji ujemn ej – pik od pow iadaj¹cy jo nowi
ben zoe sano wemu (m/z = 121). Nas têp nie anal izie pod -
dano spi ryt us ska ¿ony Bi trexem, do kt órego do dano nie -
wielk¹ ilo œæ pod chlor ynu sodu (10 µl/100 ml ska ¿onego
al koh olu). Przy jo niz acji do datn iej op rócz jonu o sto -
sunku m/z = 325 po jawi³ siê do datk owy pik o ma sie 360
(masa wi êksza o masê chlo ru). Przy jo niz acji ujemn ej
uzys kano uk³ady pików cha rakt ery sty czne dla chlorow -
co pochodnych. Anal iza ska¿ one go spi ryt usu, do któ rego
do dano pod chlor yn sodu w ilo œci po wod uj¹cej za nik piku
o cza sie re tenc ji 10,30 min na chro mat ogr amie HPLC,
wska za³a na za nik piku o m/z = 325, a wiêc od pow ia -
daj¹cemu jo nom de nat oni um. Anal iza tego roz tworu
w jo niz acji ujemn ej po twierd zi³a ob ecnoœæ chlo rowc opo -
cho dnych. Wid ma ma sowe omaw iany ch rozt worów przy
jo niz acji do datn iej przed staw iono na ry cin ie 2, na tom iast
przy jo niz acji ujemn ej – na ry cin ie 3.
4. Wy niki anal iz prób ek do wod owy ch
Oprac owa na me toda oznac zania ben zoe sanu dena -
tonium oraz wy niki prze prow adz onych eksp erym ent ów
zo sta³y wy kor zyst ane w ru tyn owej pra cy przy sporz¹dza -
niu eks pert yz. Pon i¿ej przed staw iono wy niki ba dañ otrzy-
mane w jed nej ze spraw.
Pro kur ator pro wadz¹cy spr awê po dejr zewa³, ¿e w za -
k³adzie zaj muj¹cym siê prz etwórstwem ska ¿onego al -
koh olu na stê puje usuw anie œrod ka ska¿ aj¹cego, tj. Bit -
rexu. Me chan izm tego pro cesu nie by³ zna ny. Zak³ad ku -
powa³ sk a¿o ny al koh ol, z kt óre go wy rab ia³ ró ¿ne pro -
dukty, np. p³yny an tyb akte ryj ne, p³yny do my cia szyb czy
p³yny do spry skiw ania szyb sa moc hodo wych. Do ba dañ
przes³ano p³yny oraz ko pie norm zak³ad ow ych sto sow a -
ny ch przy sporz¹dza niu pro duktów. W ta beli I ze brano
wy niku anal iz me tod¹ wy sok osp rawnej chro mat ogr afii
cie czow ej.
Jak wy nika z da nych za wart ych w tej ta beli, do wod o -
we spi ryt usy mo¿ na pod zie liæ na dwie lub trzy gru py.
W do wod ach nr 1 i 2 st ê¿e nie ben zoe sanu de nat oni um
wy znac zone na po staw ie dw óch ró ¿nych pi ków chroma -
to graficznych by³o jed nak owe (w gra nic ach b³êdu anal i -
ty cznego me tody). W do wod ach nr 3–11 stwier dzono
obecno œæ jonów ben zoe sano wych, nie stwier dzono na -
tom iast na chro mat ogr amach HPLC dru giego piku o cza -
sie re tenc ji 10,30 min. W przy padku p³ynu z do wodu
12 stê¿ enie Bi trexu ob lic zone na po staw ie za wart oœci jo -
nów ben zoe sano wych by³o wy¿ sze ni¿ uzys kane na pod -
staw ie anal izy dru giego piku, a ró¿ nica znacz nie prze -
kra cza³a b³¹d anal ity czny.
Powy ¿sze p³yny pod dano rów nie¿ ba dan iom za po -
moc¹ spek trom etru mas. W p³ynach z dowodów 1 i 2 po -
twierd zono ob ecnoœæ jo nów de nat oni um (a tym sa mym
Bi trexu). Wy niki tych ba dañ wska za³y równ ie¿, ¿e ww.
p³yny nie za wier aj¹ pr oduk tów re akc ji ben zoe sanu dena -
tonium z pod chlor ynem sodu. Stwier dzone stê¿en ia Bi t -
rexu w p³ynach z dowodów 1 i 2 by³y mniej sze ni¿
wy mag ana mi nim alna il oœæ zgod nie z roz porz¹dze niem
Mi nis tra Rol nict wa oraz Roz woju Wsi i dla tego te p³yny
zgod nie z obo wi¹zuj¹cym pra wem nie mog³y byæ zali -
czone jako sk a¿o ny al koh ol etyl owy. Z dru giej jed nak
stro ny z prak tyki In styt utu Eks pert yz S¹do wych wy nika,
¿e tech niczny 20% rozt wór Bi trexu za wiera znacz nie
mniej sze jego iloœci. Wy znac zone st ê¿en ie Bi trexu me -
tod¹ HPLC w ta kich p³ynach wy nosi³o 10–12%. P³yny
sto sow ane do ska ¿an ia s¹ ba dane przed ich u¿yciem, jed -
nak me tody anal ity czne sto sow ane w tym celu s¹ wy soce
nie spec yfi czne (naj czêœ ciej do ta kich ba dañ wykorzys -
tuje siê po miar gê stoœci). Mo ¿liwe jest za tem, ¿e ni ¿sza
za wartoœæ Bi trexu w p³ynach z dowodów 1 i 2 wy nika³a
z mniej szej jego zawar toœci w p³ynie ska¿aj¹cym.
Anal iza spek trom etr yczna p³ynów z dowo dów 3–11
po twierd zi³a, ¿e ww. p³yny nie za wier aj¹ jo nów de nat o -
ni um (a wiêc nie za wier aj¹ Bi trexu), stwier dzono nato -
miast ob ecn oœæ pi ków ma sow ych mog¹cych po cho dziæ
od pr odu któw re akc ji ben zoe sanu de nat oni um z pod -
chlor ynem sodu. Na tej pod staw ie mo¿ na przyj¹æ, ¿e
p³yny z dowodów 3–11 za wiera³y ben zoe san de nat oni um
(Bi trex), kt óry na stêpnie zo sta³ z nich us uniêty, np. po -
przez do dan ie pod chlor ynu sodu.
Anal iza prób ki p³ynu z do wodu 12 za po moc¹ spek -
trom etru mas po twierd zi³a, ¿e p³yn ten za wiera ben z o e -
san de nat oni um. W uzys kany ch wid mach ma sow ych
za rów no w jo niz acji do datn iej, jak i ujemn ej, za obs erwo -
wano do datk owe piki ma sowe po chodz¹ce prawdo po -
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
Ocena efektywnoœci metody usuwania benzoesanu denatonium... 297
dobnie od prod ukt ów re akc ji pod chlor ynu sodu z Bit -
rexem, ident yczne jak w trak cie anal izy roz tworu wzor -
cow ego ben zoe sanu de nat oni um w spi ryt usie, do któ rego
do dano pod chlor yn sodu. Po twierd zi³o to hi potezê wy -
nik aj¹c¹ z wy nik ów anal iz me tod¹ HPLC, ¿e cz êœæ jo nów
de nat oni um zo sta³o us uniêtych z p³ynu z do wodu 12,
praw dop odo bnie po przez poda nie pod chlor ynu sodu.
5. Pod sum owa nie
Wy niki prze prow adz onych badañ wska zuj¹ jedno -
znacznie, ¿e do dan ie pod chlor ynu sodu do al koh olu ety -
lowego ska¿ one go Bi trexem po wod uje cz êœc iowe lub
ca³ko wite us uni êcie tego œr odka sk a¿aj¹cego. Efek tyw -
noœæ oczyszc zania zale¿y od objêtoœ ci do dan ego pod -
chlor ynu sodu.
Oprac owa na pro ced ura ba dan ia wy rob ów spi ryt uso -
wych za po moc¹ me tody HPLC oraz spek trom etr ii mas
po zwala na wy znac zenie st ê¿en ia ben zoe sanu denato -
nium (Bi trexu) po przez oznac zenie zawar toœci jonów
ben zoe sano wych, jak rów nie¿ zaw art oœci jon ów dena -
tonium lub cz¹ste czek ben zoe sanu de nat oni um. Anal iza
zawa rto œci tych sk³adni ków po zwala us taliæ, czy ba dany
p³yn za wiera Bi trex, czy te¿ p³yn ten by³ sk a¿ony Bi t -
rexem, a na stêpnie zo sta³ od ka¿ony. In form acje te s¹ bar -
dzo przydatne przy opracowywaniu ekspertyz dla celów
s¹do wych.
Prob lems of Fo ren sic Sci ences 2005, LXIII, 288–298
298 D. Zuba, C. Œwiegoda, B. Byrska i in.
